Central to the field of microbiology is the ability to positively identify microorganisms at the level of genus, species or serotype. Correct identification is not only an essential tool in the laboratory, but it plays a significant role in the control of microbial contamination in the processing of food stuffs, the production of agricultural products, and the monitoring of environmental media such as ground water. Increasing stringency in regulations that apply to microbial contamination have resulted in a corresponding increase in industry resources which must be dedicated to contamination monitoring.
Of greatest concern is the detection and control of pathogenic microorganisms. Although a broad range of microorganisms have been classified as pathogenic, attention has primarily focused on a few bacterial groupings such as Escherichia, Salmonella, Listeria and Clostridia. Typically, pathogen identification has relied on methods for distinguishing phenotypic aspects such as growth or motility characteristics, and for immunological and serological characteristics. Selective growth procedures and immunological methods are the traditional methods of choice for bacterial identification and these can be effective for the presumptive detection of a large number of species within a particular genus. However, these methods are time consuming and are subject to error. Selective growth methods require culturing and subculturing in selective media, followed by subjective analysis by an experienced investigator. Immunological detection (e.g., ELISA) is more rapid and specific, however, it still requires growth of a significant population of organisms and isolation of the relevant antigens. For these reasons interest has turned to detection of bacterial pathogens on the basis of nucleic acid sequence.
It is well known, for example, that nucleic acid sequences associated with the ribosomes of bacteria are often highly conserved across genera and are therefore useful for identification (Webster, U.S. Pat. No. 4,717,653 and U.S. Pat. No. 5,087,558; Enns, Lab. Med., 19, 295, (1988); Mordarski, Soc. Appl. Bacteriol. Tech. Ser., 20 (Chem. Methods Bact. Syst.), 41, (1985)). Weisburg et al. (EP 51736) disclose a method for the detection and identification of pathogenic microorganisms involving the PCR amplification and labeling of a target nucleotide for hybridization to 16S rDNA of E. coli. Lane et al. (WO 9015157) teach universal nucleic acid probes that hybridize to conserved regions of 23S or 16S rRNA of eubacteria.
Although bacterial ribosomal nucleic acids contain highly conserved sequences, they are not the only sources of base sequence conservation that is useful for microorganism identification. Wheatcroft et al. (CA 2055302) describe the selection of transposable elements, flanked by unique DNA sequences, for the detection of various Rhizobium strains. Similarly, Tommassen et al. (WO 9011370) disclose polynucleotide probes and methods for the identification and detection of gram-positive bacteria. The method of Tommassen et al. relies on probes corresponding to relatively short fragments of the outer membrane protein, OmpA, which is known to be highly conserved throughout gram-positive genera. Atlas et al. (EP 517154) teach a nucleic acid hybridization method for the detection of Giardia sp. based on designing probes with sequences complementary to regions of the gene encoding the giardin protein. Webster et al. (U.S. Pat. No. 4,717,653) has expanded upon the use of rRNA in disclosing a method for the characterization of bacteria based on the comparison of the chromatographic pattern of restriction endonuclease-digested DNA from the unknown organism with equivalent chromatographic patterns of at least 2 known different organism species. The digested DNA has been hybridized or reassociated with ribosomal RNA information-containing nucleic acid from (or derived from) a known probe organism. The method of Webster et al. effectively establishes a unique bacterial nucleic acid "fingerprint" corresponding to a particular bacterial genus against which unknown "fingerprints" are compared.
Methods for the identification of Listeria monocytogenes on using specific hybridization probes or primers are known. For example, U.S. Pat. No. 5,523,205 and JP 05219997 teach DNA probes capable of hybridizing to a portion of the genome of pathogenic Listeria monocytogenes, but do not hybridize to genomes of other Listeria species. DE 4238699 and EP 576842 teach methods for detection of Listeria monocytogenes using primers designed to give amplification products specific to the monocytogenes genome. EP 576842 discusses a method for the detection of L. monocytogenes using amplification primers based on genes encoding the highly conserved iap (invasion-associated protein) of Listeria and WO 9008841 teaches nucleic acid probes capable of hybridizing to ribosomal RNA (rRNA) or rDNA of Listeria and not to rRNA or DNA of non-Listeria.
The methods described above are useful for the detection of bacteria, but each relies upon knowledge of a gene, protein, or other specific sequence known a priori to be highly conserved throughout a specific bacterial group. An alternative method would involve a nontargeted analysis of bacterial genomic DNA for specific non-phenotypic genetic markers common to all species of that bacteria. For example, genetic markers based on single point mutations may be detected by differentiating DNA banding patterns from restriction enzyme analysis. As restriction enzymes cut DNA at specific sequences, a point mutation within this site results in the loss or gain of a recognition site, giving rise in that region to restriction fragments of different length. Mutations caused by the insertion, deletion or inversion of DNA stretches will also lead to a length variation of DNA restriction fragments. Genomic restriction fragments of different lengths between genotypes can be detected on Southern blots (Southern, J. Mol. Biol. 98, 503, (1975)). The genomic DNA is typically digested with any restriction enzyme of choice, the fragments are electrophoretically separated, and then hybridized against a suitably labeled probe for detection. The sequence variation detected by this method is known as restriction length polymorphism or RFLP (Botstein et al., Am. J. Hum. Genet. 342, 314, (1980)). RFLP genetic markers are particularly useful in detecting genetic variation in phenotypically silent mutations and serve as highly accurate diagnostic tools.
Another method of identifying genetic polymorphic markers employs DNA amplification using short primers of arbitrary sequence. These primers have been termed "random amplified polymorphic DNA" or "RAPD" primers (see Williams et al., Nucl. Acids. Res., 18, 6531 (1990) and U.S. Pat. No. 5,126,239; also EP 0 543 484 A2, WO 92/07095, WO 92/07948, WO 92/14844, and WO 92/03567). The RAPD method amplifies either double or single-stranded, nontargeted, arbitrary DNA sequences using standard amplification buffers, dATP, dCTP, dGTP and TTP, and a thermostable DNA polymerase such as Taq. The nucleotide sequence of the primers is typically about 9 to 13 bases in length, between 50 and 80% G+C in composition and contains no palindromic sequences. RAPD detection of genetic polymorphisms represents an advance over RFLP in that it is less time consuming, more informative, and readily susceptible to automation. Because of its sensitivity for the detection of polymorphisms, RAPD analysis and variations based on RAPD/PCR methods have become the methods of choice for analyzing genetic variation within species or closely related genera, both in the animal and plant kingdoms. For example, Landry et al. (Genome, 36, 580, (1993)) discuss the use of RAPD analysis to distinguish various species of minute parasitic wasps that are not morphologically distinct. Van Belkum et al. (Mol. Biochem Parasitol., 61, 69, (1993)) teach the use of PCR-RAPD for the distinction of various species of Giardi.
In commonly assigned U.S. Pat. No. 5,340,728, Applicants disclosed a method of double-nested PCR which is used to detect the presence of a specific microbe. This disclosure first describes identifying a random, unique segment of DNA for each individual microorganism which will be diagnostic for that microorganism. To identify and obtain this diagnostic nucleic acid segment a series of polymorphic markers is generated from each organism of interest using single primer RAPD analysis. The RAPD series from each organism is compared to similarly generated RAPD series for other organisms, and a RAPD marker unique to all members of the group is then selected. The unique marker is then isolated, amplified and sequenced. Outer primers and inner primers suitable for double-nested PCR of each marker may then be developed. These primers comprise sequence segments within the RAPD markers, wherein the inner set of primers will be complementary to the 3' ends of the target piece of nucleic acid. These nested primers may then be used for nested PCR amplification to definitely detect the presence of a specific microorganism.
In commonly owned PCT U.S. 95/06704 (WO 95/33854), Applicants more particularly adapted and described this RAPD methodology to identify a sequence or marker. The presence of the marker is diagnostic for all individuals of the genus Salmonella. PCT U.S. 95/06704 teaches a method involving a RAPD amplification of genomic DNA of a representative number of Salmonella individuals to produce a RAPD amplification product, termed the diagnostic fragment. This diagnostic fragment must be present in the RAPD profiles in over 90% of the individuals tested. Sequence information from the diagnostic fragment enables identification of the most suitable PCR primer binding sites within the diagnostic fragment to define a unique diagnostic marker. Primers flanking this marker are useful for the generation of amplification products from Salmonella genomic DNA, but will not produce any amplification products in non-Salmonella genera.
In commonly owned U.S. Ser. No. 08/608,881, Applicants disclose a method, diagnostic sequences and primers that are useful in the identification of the Escherichia coli 0157:H7 serotype. The method involves the identification of a RAPD-amplified DNA fragment common to 0157:H7 Escherichia coli, the identification of the most conserved regions of that fragment, and the preparation of specific primers useful for detecting the presence of a marker within the fragment whereby that set of primers is then useful in the identification of all 0157:H7 Escherichia coli. The method of 08/608,881 does not teach markers useful for the specific identification of Listeria monocytogenes and Listeria spp.
A detection methodology using PCR/RAPD specific to Listeria monocytogenes and Listeria spp. would be of high utility in the food industry. Detection methods not dependent on sequences derived from a known gene or associated with a known phenotypic characteristic of Listeria monocytogenes and Listeria spp. have not previously been disclosed.